Intercellular chemical signaling, also known as quorum sensing, is widely used by bacteria to control gene expression, protein activity, and behaviors in a coordinated manner between individual cells within a community. Gram-positive bacteria, including the important human pathogen Streptococcus pneumoniae, utilize secreted peptides as intercellular signals, referred to here as ?pheromones?. The Rap/Rgg/Npr/PlcR/ PrgX (RRNPP) family of quorum sensing systems are widespread among Gram-positive bacteria, and within individual genomes, multiple paralogs of pheromone receptors are typically found. The clear majority of these systems are unstudied (9 systems are predicted in S. pneumoniae strain D39), largely due to the current limitations on the ability to identify pheromone genes and their regulatory targets. RRNPP peptide pheromones are encoded by small open reading frames (sORFs), which, due to their small size and lack of unique characteristics, are generally unrecognizable in genome sequences. This proposal outlines the development of systematic genome-wide analyses that will identify sORFs encoding RRNPP pheromones (as well as all other unannotated sORFs that may have important physiological activities) and the regulons of RRNPP quorum sensing systems. To accomplish these goals, RNA-seq analysis, combined with the cutting-edge approach of antibiotic-assisted ribosome profiling (Ribo-seq), will be performed to directly identify and enumerate all transcription and translation events in bacterial cultures. Development of these methodologies will allow for a rapid increase in the ability to study communication systems in bacteria and will likely accelerate efforts to manipulate signaling pathways as a means to alter microbial behaviors, especially virulence attributes of pathogens.